Validation of selected candidate proteins by fluorescence microscopy.

<p>(<b>A</b>) Live cell imaging of cells expressing Shs1-mCherry and Bni4-GFP at indicated time points of the cell cycle. Scale bar 5 μm. (<b>B</b>) Fluorescence microscopy images of blocked cells expressing Shs1-mCherry and Nip1-, Pno1-or Tub1-GFP. Scale bar 6 μm.</p

Timing of the interaction of Bud3 and Bud4 with the septins.

<p><b>(A)</b> Time lapse microscopy of Bud3-GFP or Bud4-GFP and Shs1-mCherry expressing cells. The time of the first appearance of the GFP fusion protein after the appearance of Shs1-mCherry at the bud neck was recorded. <b>(B)</b> Pulldown of Bud3-TAP and Bud4-TAP on immobilized septin rods and detection with an anti-Protein A antibody. The asterisk marks the hardly visible Bud3-TAP signal. A cross reaction of the antibody with one of the septins is marked with +. <b>(C)</b> Time resolved SPLIFF analysis (N_ub-Bud3 or N_ub-Bud4 vs. Shs1-CCG). Imaging time frame 3 min, 100 μM Cu²⁺. N = 10 for Bud3 and N = 9 for Bud4. The start of the N_ub induced conversion of Shs1-CCG is marked with an arrow.</p

SILAC-AP-MS analysis.

<p>Results of the quantitative MS-analysis. Volcano plots for the MS analysis of cells arrested in G1 phase (alpha facor). (<b>A</b>), S-phase (<b>B</b>) or anaphase (<b>C</b>). The red-dashed lines indicate the thresholds defining a specific interaction. Selected candidate proteins for further validation are named and labeled in blue, septins are marked in red. Specific interactors were grouped into the indicated categories (<b>D</b>). Numbers represent the specific interactors in the respective timepoints for each category.</p

Workflow for SILAC-AP-MS.

<p>Yeast strains expressing Cdc11-TAP and Cdc11-GFP (metabolically labelled with ¹³C₆,¹⁵N₄-L-arginine and ¹³C₆,¹⁵N₂-L-lysine) are blocked with the indicated methods and subjected separately to affinity purification. The eluates are mixed and subjected to separation by SDS-PAGE followed by nano HPLC-ESI-MS/MS analysis and statistical data evaluation.</p

Overview of the SILAC-AP-MS datasets.

<p><b>(A)</b> Overlap of the specific hits in alpha-factor arrested cells, S-phase (HU) and late anaphase (<i>cdc15-1</i>). <b>(B)</b> Summary and overlap of the candidate proteins selected for further validation.</p

Steady-state SPLIFF measurements.

<p>Each point represents a single cell measurement. The expressions of the N_ub -fusion proteins were induced by100 μM Cu²⁺. The calculated medians and SEMs are shown in red. N_ub-fusions are grouped according to their interaction signal. (<b>A</b>) Strong interaction (SPLIFF signal ≥ 70%). (<b>B</b>) Weak interaction (SPLIFF signal ≥ 30%). (<b>C)</b> No interaction (SPLIFF signal ≤ 30%). The negative value for Nap1 might result from a weaker expression of Nap1 than the control N_ub. Note a subpopulation of Glc7 cells that have a SPLIFF signal >50%. grey squares: negative control N_ub-empty; grey circle: positive control N_ub-Cdc11. (<b>D</b>) Comparison between stable collar and split septin rings SPLIFF signals induced by N_ub- and -Bud4 with 0 (left) or 100 μM Cu²⁺ (right).</p

SPLIFF- and alternative evaluation of 26 selected candidates.

<p>SPLIFF- and alternative evaluation of 26 selected candidates.</p

Cell synchronization of Cdc11-GFP and Cdc11-TAP expressing cells.

<p>Representative image sections (z-projection) for Cdc11-GFP or Cdc11-TAP expressing strains using the indicated blocking approaches. The structure within the white rectangle represents a zoom in of the respective septin architecture using a 100x objective. White arrowheads indicate split septin rings in <i>cdc15-1</i>-arrested cells. Scale bar 6 <b>μ</b>m.</p